10 11 12 13 14 15 16 17 18 · 2020. 6. 28. · 9 1Department of Otolaryngology, University of California, San Fr ancisco, CA, USA 10 2Department of Pharmaceutical Chemistry, University

1 Size-dependent protein segregation creates a spatial switch for Notch and APP signaling 2 3 Minsuk Kwak1,2,3,4,5,6,*, Kaden M. Southard1,2,*, Nam Hyeong Kim1,2,3,6, Ramu Gopalappa4,8, Woon 4 Ryoung Kim1,2,3, Minji An4,5, Hyun Jung Lee1,2,3, Justin Farlow2, Anastasios Georgakopoulos9, Nikolaos 5 K. Robakis9, Daeha Seo10, Hyeong Bum Kim4,5,8,11,12, Yong Ho Kim6,7, Jinwoo Cheon4,5,13, Zev J. 6 Gartner2,14,† , Young-wook Jun1,2,3,4,5† 7 8 1Department of Otolaryngology, University of California, San Francisco, CA, USA 9 2Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA 10 3Helen Diller Family Cancer Comprehensive Center (HDFCCC), University of California, San Francisco, 11 CA, USA 12 4Center for Nanomedicine, Institute for Basic Science (IBS), Seoul, Republic of Korea 13 5Graduate Program of Nano Biomedical Engineering (Nano BME), Advanced Science Institute, Yonsei 14 University, Seoul, Republic of Korea 15 6SKKU Advanced Institute of Nanotechnology (SAINT), Sungkyunkwan University, Suwon, Republic of 16 Korea 17 7Department of Biomedical Engineering, Sungkyunkwan University, Suwon, Republic of Korea 18 8Department of Pharmacology, Yonsei University College of Medicine, Seoul, Republic of Korea 19 9Department of Psychiatry and Neuroscience, Icahn School of Medicine at Mount Sinai, New York, 20 USA 21 10Department of Emerging Materials Science, DGIST, Daegu, Republic of Korea 22 11Brain Korea 21 Plus Project, Yonsei University College of Medicine, Seoul, Republic of Korea 23 12Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Republic of 24 Korea 25 13Department of Chemistry, Yonsei University, Seoul, Republic of Korea 26 14Chan Zuckerberg Biohub, University of California San Francisco, CA, USA 27 28 *These authors contributed equally. 29 †Correspondence should be addressed to Z.J.G. ([email protected]) or Y.J. (young-30 [email protected]). 31

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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted June 29, 2020. ; https://doi.org/10.1101/2020.06.28.176560doi: bioRxiv preprint

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Aberrant cleavage of Notch and amyloid precursor protein (APP) by gg-secretase is implicated in 33 developmental disorders, cancer, and dementia. How cleavage is regulated in space and time is 34 unclear. Here, we report that size-dependent protein segregation acts as a spatial switch 35 regulating the dynamic distribution of these receptors relative to their enzymatic partner, gg-36 secretase. Because the size of the extracellular domain (ECD) greatly exceeds the width of the 37 junctional cleft, Notch is excluded from cadherin-based adherens junctions (cadAJs) where gg-38 secretase localizes, prohibiting the enzyme-substrate interaction. ECD elimination by ADAM 10/17 39 drives a spatial rearrangement of Notch into cadAJs where gg-secretase cleavage occurs. Spatial 40 mutation of Notch by decreasing (or increasing) the ECD size promotes (or inhibits) downstream 41 signaling, respectively, regardless of ligand-presentation or S2 cleavage. Elimination of cadAJs 42 abolishes Notch activation, confirming the indispensable role of cadAJs in regulating Notch-gg-43 secretase interactions. We further show that this spatial switch regulates APP cleavage and 44 amyloid beta production. Therefore, size-dependent protein segregation creates membrane 45 domains with unique proteolytic activities, choreographing the proteolytic sequence while 46 suppressing nonspecific signal activation. 47

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Notch is a highly conserved mediator for contact-dependent cell-cell communication, which orchestrates 49 diverse functions in metazoans1-5. Tight control of Notch signal activation is essential for many 50 developmental processes, while dysregulation of Notch activation can cause severe disease including 51 developmental, neurological, and immunological disorders and cancer1-8. Accordingly, to enable precise 52 signal regulation, receptor activation occurs through multiple steps, independently gated by chemical 53 (e.g., ligand-receptor interactions, posttranslational modifications) and mechanical cues4,5,9-27. However, 54 many signaling processes involving physical contact between two cells – so called juxtacrine signaling 55 processes – are also regulated by spatial cues28-31. As exemplified by the kinetic segregation model in 56 immune cells28,32-43, spatial rearrangements of these signaling molecules modify their physical and 57 biochemical environments to facilitate receptor activation. Notch, which also mediates signal exchange 58 by physical contact, is subjected to similar structural and spatial rearrangements44,45. To test whether 59 Notch signaling is regulated by these spatial rearrangements, we investigated the spatial dynamics of 60 Notch and its signaling partners at cellular interfaces. Specifically, we focused on four key signaling 61 molecules – Notch, delta-like ligand 1 (Dll1), ADAM 10, and g-secretase, relative to cadherin-based 62 adherens junctions (cadAJs); the cell-cell adhesions that initiate many structural and spatial changes by 63 bringing and holding the membranes together46-49 (Fig. 1a). 64

CadAJs segregate Notch from gg-secretase, preventing interactions 65 To map the distribution of Notch signaling components, we generated a series of U2OS cells expressing 66 recombinant Notch1, Dll1, and/or epithelial cadherin (E-cadherin, Ecad) proteins. To facilitate the imaging 67 of these proteins, we fused them with self-labeling tags (SNAP or Halo-tags) and/or fluorescent proteins 68 (EGFP or mCherry) at the N- or C-termini, respectively (Extended Data Table 1). The endogenous Notch 69

processing enzymes (i.e. ADAM 10, and g-secretase) were immunostained and then imaged by confocal 70 microscopy. With the exception of ADAM 10 which exhibited no preferential distribution relative to cadAJs 71 (Extended Data Fig. 1a), all other proteins exhibited cadAJ-dependent localization (Fig. 1b, c). g-72 secretase (visualized by staining with an anti-presenilin-1 antibody) was strongly enriched at the cadAJs 73 with negligible non-junctional membrane signal (Fig. 1b, c, Extended Data Fig. 1b, c)50. In contrast, both 74 Notch and Dll1 were excluded from cadAJs (Fig. 1b, c) and consequentially g-secretase (Extended Data 75 Fig. 2a, b). Notch exclusion from cadAJs was observed in multiple contexts, including different cadherin 76 types, cell types, and cell polarization states (Extended Data Fig. 3). Quantitative analysis using the 77 Manders’ overlap coefficient to calculate fractional overlap with E-cadherin also confirmed the enrichment 78

of g-secretase (0.85 ± 0.21) at cadAJs and the exclusion of Notch (0.24 ± 0.19) and Dll1 (0.26 ± 0.18) 79 from cadAJs (Extended Data Fig. 2c, d). These observations suggest two mechanisms by which cadAJs 80 might influence Notch signaling: first, cadAJs recruit g-secretase; second, cadAJs segregate Notch 81

ligands and receptors from g-secretase to prevent their interactions. 82

To interrogate how cadAJs drive the spatial segregation of the enzyme (i.e. g-secretase) and 83 substrate (i.e. Notch) pair, we employed our recently developed single-cell perturbation tool, 84


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mechanogenetics, where magnetofluorescent nanoparticles (MFNs) control the location and mechanical 85 loading of targeted receptors hence cell signaling26,27,51. Specifically, we clustered E-cadherin using 86 mechanogenetics to generate artificial cadAJs that recapitulate the functional and signaling roles of native 87 cadAJs26,27,51 (Fig. 1d, e; See methods for more details). We then monitored the consequence of cadAJ 88 formation on the spatial distributions of g-secretase, Notch, and associated proteins (e.g. Flotillin-1, Flot1) 89

relative to the artificial cadAJ (Fig. 1f, g). Consistent with the native cell-cell cadAJs, g-secretase was 90 localized at the artificial cadAJ (Fig. 1f and Extended Data Fig. 4a, b). We also observed colocalization 91 of Flot1, a protein enriched in spatially discrete and ordered membrane microdomains at the artificial 92 cadAJs52-56 (Fig. 1f and Extended Data Fig. 4a, b). This observation, along with analysis of native cell-93 cell junctions (Extended Data Fig. 4, c-g), molecular dynamics (MD) simulation (Extended Data Fig. 4, 94 h-j), and cholesterol depletion experiments (Extended data Figs. 4k), suggests that the cadAJs recruit 95 and stabilize g-secretase through a common spatially discrete and ordered membrane microdomains (for 96 more discussion, see Supplementary Discussion). 97

Surprisingly, we observed an intense Notch signal at the artificial cadAJ (Fig. 1g and Extended 98 Data Fig. 5), in contrast to the depleted Notch signal at native cell-cell cadAJs (Fig. 1, a-c). The 99 extraordinarily large size (extended length = 136 nm12, compacted height by electron microscopy > 20 100 nm57) of the Notch extracellular domain (NECD) suggested a potential explanation for these contradicting 101 observations. Specifically, Notch could be excluded from cadAJs due to the NECD size greatly exceeding 102 the narrow intermembrane cleft created by native cadAJs (20 nm)47,58. On the other hand, artificial cadAJs 103 generated by MFNs are free of membrane juxtaposition and hence allow for Notch diffusion and 104 accumulation, presumably through its association with γ-secretase or other components of the membrane 105 microdomains59. These observations fit a model wherein the size-based physical constraint caused by 106 cell-cell cadAJs segregates Notch from γ-secretase, preventing interactions that would otherwise serve 107 to colocalize and concentrate the enzyme-substrate pair. 108

Size-dependent segregation choreographs the Notch proteolytic sequence 109 Upon activation by juxtacrine ligand-receptor interactions, Notch is processed by ADAM 10/17 (S2 110 cleavage) and then by γ-secretase (S3 cleavage), sequentially1-5. Since each of proteolytic events leads 111 to a dramatic reduction in the size of NECD, we investigated how the size changes during activation 112 correlate with its spatial distribution. We plated U2OS cells co-expressing SNAP-NFL-mCherry and Ecad-113 GFP on a Dll4-coated substrate to trigger Notch activation. Cells were also treated with TAPI2 (an ADAM 114 10/17 inhibitor which stabilizes full-length Notch) or DAPT (a γ-secretase inhibitor that stabilizes the S2-115 cleaved Notch having a minimal extracellular domain) to capture Notch intermediates (Fig. 2a). To 116 quantify the spatial redistribution of each Notch intermediate relative to the cadAJ, we measured the 117 average mCherry fluorescence signal inside (IIN) and outside (IOUT) of the cadAJ and estimated an 118 enrichment ratio (IIN/IOUT, See Methods). An intensity ratio of 1 represents no cadAJ-dependent 119 distribution as validated with Dil dyes (IIN/IOUT = 1.07, Fig. 2c and Extended Data 6a), and values less 120


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than or greater than 1 represent exclusion from or enrichment within cadAJs, respectively. With TAPI2, 121 we observed minimal mCherry fluorescence signal from cadAJs (IIN/IOUT = 0.50, Fig. 2b(ii), c(ii)), 122 indicating that the ligand-receptor interaction did not alter the spatial distribution of Notch before S2 123 cleavage. In contrast, when TAPI2 was removed to activate S2 cleavage and DAPT was added to inhibit 124 S3 cleavage, we observed strong enrichment of the mCherry signal at cadAJs (IIN/IOUT = 2.14, Fig. 2b(iii), 125 c(iii)), indicative of relocalization and concentration of the Notch extracellular domain truncation (NEXT), 126 the product of S2 cleavage, into cadAJs. When 𝛾𝛾-secretase activity was rescued by washing out DAPT, 127 the initially intense mCherry signal at cadAJs gradually disappeared (Fig. 2b(iv), c(iv), and Extended 128 Data Fig. 6b, c), presumably corresponding to release of Notch intracellular domain (NICD) from the 129 membrane. These results suggest a role for the size-dependent protein segregation as a spatial switch 130 that regulates the distribution of Notch intermediates, thereby choreographing the sequential steps in 131 Notch proteolysis. According to this model, the mass of NECD poses a physical constraint preventing 132 entry of Notch to the narrow space between membranes in the cadAJ cleft where 𝛾𝛾-secretase is localized. 133 Hence, 𝛾𝛾-secretase cannot process the full-length Notch before S2 cleavage. Following S2 cleavage, 134 removal of NECD relieves the physical constraint, allowing Notch to enter into the cadAJ cleft. This 135 facilitates a productive Notch-𝛾𝛾-secretase interaction59, S3 cleavage, and then downstream signaling. 136 To explore how size-dependent segregation controls Notch signaling, we first generated a series 137 of U2OS cell lines stably expressing Notch variants with different truncation lengths: a partial deletion of 138 the EGF repeats (NΔEGF1-25, approximate height: 48 nm), complete deletion of the EGF repeats but 139 retention of negative regulatory region (NΔEGF, approximate height: 10 nm), and a complete removal of 140 NECD (NEXT, approximate height: 4 nm) (Fig. 2d). We fused these Notch variants with SNAP- and 141 mCherry-tags at their N- and C-termini, to differentially image the extracellular and intracellular domains. 142 All cells were treated with TAPI2 and DAPT to prevent any potential proteolysis of the variants. Consistent 143 with predictions based on size-dependent protein segregation, NΔEGF1-25, the Notch variant with an ECD 144 taller than the height of the intermembrane cadAJ cleft, was excluded from cadAJs (IIN/IOUT = 0.57) (Fig. 145 2e, f). NEXT with an ECD smaller than the junctional height was enriched at cadAJs (IIN/IOUT = 2.39) (Fig. 146 2e, f). Interestingly, we observed binary localizations of NΔEGF (intermediate height) relative to cadAJs, 147 with a mean IIN/IOUT value of 1.32 (Fig. 2e, f). Some cadAJs displayed NΔEGF enrichment (Fig. 2e(bottom 148 left), f(right)), consistent with the size-based prediction. Meanwhile, other cadAJs excluded NΔEGF (Fig. 149 2e(bottom right), f(right)). Considering the fact that cadherin cluster densities within cadAJs vary with the 150 size, type, and degree of junction maturation60,61 and that the glycosylated negative regulatory region 151 domain62 is susceptible to steric crowding, this unanticipated exclusion might result from a lateral 152 crowding effect in high-density cadAJs. 153

We then investigated the functional consequences of NΔEGF relocalization to cadAJs by 154 measuring the impact on downstream signaling process. Focusing on the subset of cadAJs showing 155 strong enrichment for Notch signal, we inferred γ-secretase activity by measuring the changes in 156 extracellular SNAP (labeled with SNAP surface dye) and intracellular mCherry fluorescence signals of 157


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NΔEGF after DAPT removal while maintaining TAPI2 concentration. This construct has different 158 fluorescence markers on the extracellular and intracellular domains of the protein, allowing us to 159 differentially map the S2 and S3 cleavage events of the construct. While SNAP fluorescence signal 160 remained strong, the mCherry signal at the cadAJ was negligible (Fig. 2g). Similarly, colocalization 161 analysis of multiple cadAJs using Pearson correlation coefficients showed more significant signal 162 reduction in the mCherry channel than in SNAP, indicating selective release of NICD from cadAJs (Fig. 163 2h). We measured the kinetics of S3 processing by tracking mCherry intensity at the cadAJ in single cells. 164 Initially intense mCherry signals (average IIN/IOUT = 2.89 ± 1.15) rapidly dissipated within the first 2 hours 165 following DAPT removal (average IIN/IOUT = 0.81 ± 0.42), reaching a plateau at 4 hours (IIN/IOUT = 0.65 ± 166 0.14) (Fig. 2i, j, and Extended Data Fig. 6d, e). Removing DAPT did not elicit a significant reduction in 167 mCherry signal intensity from the non-cadAJ membrane, indicating that the S3 cleavage activity was 168 strongly localized at the cadAJ (Extended Data Fig. 6f). To confirm that the observed decrease in 169 mCherry signal at the cadAJ corresponds to the S3 cleavage, we performed western blot analysis of the 170 cells expressing the Notch variants. We cultured cells with TAPI2 to decouple 𝛾𝛾-secretase processing 171 from S2 cleavage, and measured cleaved NICD levels by immunoblotting with Notch anti-V1744 172 antibodies. Cells expressing NFL or NΔEGF1-25 resulted in no or minimal NICD, respectively (Fig. 2k, l). 173 Whereas, cells expressing NΔEGF produced a significant amount of NICD (Fig. 2k, l). Cells expressing 174 NEXT exhibited the highest NICD production, about a 4-fold increase compared with that of NΔEGF (Fig. 175 2k, l). The observed NICD production levels were proportional to the enrichment ratio (IIN/IOUT) of the 176 Notch variants at cadAJs, suggesting the essential role of size-dependent protein segregation as a spatial 177 switch that controls Notch activation. Additionally, the substantial NICD production from the cells 178 expressing NΔEGF indicates that, when localized together, γ-secretase can process Notch, bypassing 179 S2 cleavage. Ligand-independent activation of Notch receptors with an intact S2 site was observed 180 previously in Notch truncation variants or synNotch constructs63-65, but the mechanism of this activation 181 has been unclear so far. Our observations support the notion that colocalization of Notch with 𝛾𝛾-secretase 182 is sufficient to trigger S3 proteolysis and signaling. 183

Spatial mutation alters Notch signaling 184 Prevailing models of Notch proteolysis by γ-secretase are based on the notion that S2-cleavage of Notch 185 serves to potentiate the cleavage by modifying the molecular interface at the enzyme-substrate pair66,67. 186 For example, it has been suggested that γ-secretase selectively recognizes S2-cleaved Notch (i.e., NEXT) 187 through hydrogen bonding between a glutamate residue in nicastrin and the new N-terminus of NEXT66. 188 Another model proposed that S2 cleavage serves to reduce steric repulsion between nicastrin and NECD, 189 strengthening their interaction67. However, another key feature of S2 cleavage is that it generates a 190 smaller molecular intermediate that can uniquely access cadAJs, thereby colocalizing Notch with γ-191 secretase and significantly increasing its concentration near the enzyme active site. This suggests a third 192


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model, wherein γ-secretase activity on full length Notch and its intermediates is blocked by maintaining 193 Notch concentrations below the KM for γ-secretase through spatial segregation. 194

To explicitly test the effect of Notch spatial localization relative to cadAJs and γ-secretase on the 195 signaling, we designed two experiments that induce spatial mutation of Notch. First, we employed a DNA-196 mediated crosslinking strategy to enhance NΔEGF – a Notch variant that exhibited binary localizations 197 relative to cadAJs and relatively low Notch activation – enrichment at the cadAJ. We generated cells co-198 expressing SNAP-NΔEGF-mCherry and Halo-Ecad-GFP, and treated the cells with complementary BG- 199 and chloroalkane-modified oligonucleotides in the presence of TAPI2 and DAPT (Fig. 3a). Notch-E-200 cadherin heterodimers were formed efficiently as evidenced by the appearance of a higher molecular 201 weight band corresponding to the DNA-linked complex on western blots (Extended Data Fig. 7a). 202 Compared to untreated cells (IIN/IOUT = 1.32 ± 1.06), we observed further enrichment of NΔEGF at cadAJs 203 in the presence of DNA crosslinking (IIN/IOUT = 1.89 ± 0.91) (Fig. 3b). We then maintained cells in TAPI2 204 but removed DAPT to allow S3 cleavage. We observed decreases in mCherry signal at cadAJs after 205 DAPT removal, indicating efficient S3 cleavage without S2 cleavage (Extended Data Fig. 7b-d). 206 Interestingly, in western blots, we observed increased V1744-terminated NICD levels from the cells 207 treated with DNA crosslinkers, compared with the untreated control (Fig. 3c). There results suggest that 208 new molecular interfaces produced by mechanical activation and S2 cleavage are not necessary when 209 γ-secretase is concentrated with its substrate. Considering that DNA crosslinking (molecular weight = 210 21.4 kD) increases the ECD size of NΔEGF, the observed increase in NICD production cannot be 211 explained by the nicastrin-induced steric repulsion model. Hence, we concluded the result that increased 212 concentration of the NΔEGF at cadAJs facilitated its interaction with 𝛾𝛾-secretase and promoted S3 213 cleavage. 214 To further test the importance of size-dependent spatial segregation, we induced spatial mutation 215 of NEXT (i.e. the Notch variant that showed the strongest enrichment at the cadAJ and activation) by 216 chemically conjugating it with macromolecules. Specifically, we conjugated BG-modified polymers and 217 proteins, to the extracellular SNAP tag (4.0 nm) of the variant via BG-SNAP chemistry (Fig. 3d). Grafting 218 of these macromolecular pendants onto NEXT increases the size of the Notch construct but does not 219 modify the N-terminal amine accessibility for hydrogen bonding with nicastrin. To interrogate the size-220 dependent spatial mutation of NEXT systematically, we used a series of pendants with different 221 hydrodynamic sizes, that includes polyethylene glycol with an average molecular weight of 3.4 kD 222 (PEG3.4k, 2.5 nm), branched PEG20k (bPEG20k, 4.0 nm), linear PEG20k (ℓPEG20k, 8.0 nm), DNA-223 streptavidin conjugates (DNA-stv, 9.5 nm), and human immunoglobulin G (hIgG, 12 nm) (Fig. 3d and 224 Extended Data Fig. 8a). In the presence of DAPT, we observed a size-dependent distribution of NEXT 225 at the cadAJ, where the larger pendant resulted in a greater decrease in mCherry signal at the cadAJ. 226 With pendants smaller than 5 nm (i.e. PEG3.4k and bPEG20k), NEXT remained enriched at the cadAJ 227 with IIN/IOUT of 2.21 and 2.01, respectively (Fig. 3d, e). When ℓPEG20k, DNA-stv, or hIgG were added, 228 we observed binary localizations of NEXT that were enriched at or excluded from the cadAJs, with mean 229


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IIN/IOUT values of 1.06, 0.82, 0.98, respectively (Fig. 3d, e). These observations were similar to the spatial 230 behaviors of NΔEGF having a comparable ECD size, where only less dense cadAJs allowed Notch 231 colocalization. We then examined the signaling consequences of each size-dependent spatial mutation 232 of NEXT. Following S3 cleavage, NICD traffics to the nucleus, allowing us to measure nuclear mCherry 233 signal as a proxy for Notch pathway activation. The PEG3.4k or bPEG20k addition did not significantly 234 alter nuclear mCherry signal of NEXT, compared with cells with no pendant addition (Fig. 3f, g and 235 Extended Fig. 9b). Conjugation of ℓPEG20k, DNA-stv (9.5 nm) resulted in substantial decrease in 236 nuclear mCherry signal to 0.39 and 0.37, respectively (Fig. 3f, g and Extended Fig. 8b). hIgG addition 237 slightly further suppressed nuclear mCherry signal to 0.27 (Fig. 3f, g and Extended Fig. 8b). Finally, we 238 summarized the S3-cleaved NICD production of all Notch variants as a function of the Notch enrichment 239 factor, IIN/IOUT, in Fig. 3h, clearly visualizing the spatial dependence of S3 cleavage. 240

The cadAJ-mediated spatial switch is indispensable for Notch signaling 241 Given the significant role of cadAJs in coordinating the spatial dynamics of Notch and γ-secretase, we 242 next interrogated Notch signal activation in cells lacking cadAJs. To minimize physical contact between 243 cells and hence cadAJ formation, we sparsely plated cells expressing SNAP-Notch-Gal4 (SNAP-NFL-244 Gal4) receptors on a Dll4-coated substrate. We used a UAS-Gal4 reporter system that detects Notch 245 activation with nuclear mCherry signal15,23,26. After 16 hours from cell seeding, we measured the mCherry 246 fluorescence in cells having no prior contact with other cells. For comparison, we also analyzed the 247 mCherry signal in cells with robust cadAJs within high-density culture. While the cells with physical 248 contacts with other cells exhibited robust increase in nuclear mCherry fluorescence signal, those without 249 cell-cell contact elicited no increase in signal (Fig. 4a, b and Supplementary Video 1). Reestablishing 250 the cadAJs by plating cells on a substrate coated with Ecad-Fc and Dll4-Fc rescued the Notch signaling 251 of the solitary cells (Fig. 4a, c, and Supplementary Video 2). These results support that cadAJs are 252 required for Notch processing at the cell surface and downstream signaling. Critically, E-cadherin seems 253 to function in this capacity in a manner that is independent of its role in mediating cell-cell contact. To 254 further test this notion, we knocked out the gene encoding E-cadherin (CDH1) in the reporter cell line via 255 CRISPR-Cas9, then plated the cells at high density on Dll4-Fc coated plates (Extended Data Fig 9, a-256 f). Strikingly, E-cadherin knockout (Ecad-KO) resulted in abrogation of Notch activation even with robust 257 cell-cell contact (Fig. 4d, e, and Extended Data Fig. 9g). Reintroduction of plasmids encoding E-258 cadherin or N-cadherin into Ecad-KO cells recovered Notch activation with substantial nuclear mCherry 259 signal, confirming the indispensable role of cadAJs in Notch signaling (Fig. 4d, e and Extended Data 260 Fig. 9g). 261

Size-dependent spatial dynamics and proteolysis of amyloid precursor proteins 262 The proteolysis processes of amyloid precursor protein (APP) plays a central role in amyloid beta (Aβ) 263 pathology, which can cause failures in many organs such as brain, heart, kidney, and vasculatures68-71. 264 Interestingly, APP has a strikingly similar topology and proteolytic cleavage sequence to that of Notch. 265


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APP possesses a large ECD (> 20 nm) and is processed by a- or β-secretase and then g-secretase68-71. 266 These characteristics motivated us to investigate the role of the size-dependent protein segregation in 267 APP processing. We generated U2OS cells co-expressing APP-GFP and SNAP-N-cadherin (SNAP-268 Ncad) and monitored the cell surface spatial dynamics of APP intermediates relative to N-cadherin based 269 AJs (NcadAJs) using proteolytic enzyme inhibitors. Similar to Notch, we found that APP was initially 270 excluded from NcadAJs, diffused into the NcadAJs after the ECD shedding by a- or β-secretase, and 271

then was processed by g-secretase within it (Fig. 4f). 272

APP can be processed along two pathways by g-secretase – one pathway occurs at the cell 273 surface to generate more soluble isoforms including p3 or Aβ40, the other pathway occurs internally to 274 produce less soluble and pathogenic Aβ4268-73. Given our previous observation that loss of cadAJs leads 275 to a decrease in cell surface g-secretase, we hypothesized that APP processing would be biased under 276 these conditions towards Aβ42. We tested this hypothesis by constructing U2OS cell lines lacking both E- 277 and N-cadherins (cad-KO cells) using CRISPR-Cas9 (Extended Data Fig. 9h, i). We then transfected 278 plain U2OS cells or cad-KO cells with a plasmid encoding APP sequence and measured Aβ production 279 by ELISA. Consistent with the hypothesis, cad-KO cells produced higher Aβ42, the isoform prone to 280 severe fibril aggregation, compared to cells with endogenous cadherin expression (Fig. 4g). 281

Discussion 282 Notch displays several unique features that are distinct from other juxtacrine receptors. For example, 283 unlike classical receptor signaling, the Notch ligand-receptor interaction (chemical switch) is converted 284 into intracellular signaling through a sequence of additional signaling switches acting by mechanical, 285 enzymatic, and spatial mechanisms. These include unfolding of the negative regulatory region domain 286 (mechanical switch), S2- and S3- cleavages (enzymatic switch), and finally translocation of the NICD 287 from the cell membrane to the nucleus (spatial switch)4,5. Our study reveals that Notch additionally 288 integrates a spatial switch at the cell surface to tightly choreograph the enzymatic cleavage sequence 289 prior to NICD release. Previously, it was thought that this enzymatic sequence was regulated by 290 modification of the molecular interface between Notch and nicastrin after S2-cleavage66,67. Our model 291 does not rule out the contribution of the nicastrin-Notch chemical interface during this process, but our 292 study strongly suggests that the spatial switch is the major regulator of Notch-g-secretase interaction and 293

signaling, functioning by increasing the concentration of the g-secretase substrate to the point that it is 294 efficiently processed by the enzyme. Particularly, our mechanogenetic and the NΔEGF cleavage 295 experiment shown in Figure 1(d-g) and 2(e-l), respectively, support the notion that Notch with an intact 296 S2 site is effectively engaged and then processed by g-secretase when the spatial constraints are 297 removed. 298 The operating principle of the spatial switch is closely related to another unique feature of Notch 299 receptor: the unusually tall NECD. The functional residues responsible for ligand binding are located near 300 the N-terminus, which protrudes above the crowded cell surface, where they are poised to engage ligands 301


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on neighboring cells. Surprisingly, however, it has been also shown that replacing the EGF-like domain 302 repeats with a relatively smaller ligand binding domain (e.g. synNotch) maintains the receptor function74,75. 303 Why then does Notch receptor bear such a massive ECD? Our study provides insight into this question, 304 where the large ECD is crucial for its spatial segregation from g-secretase thereby minimizing nonspecific 305 ligand-independent activation. Low level NICD production was observed even for the Notch variants with 306 a partial EGF truncation (NΔEGF1-25), and levels gradually increased upon successive truncations to the 307 NECD size. Our model also explains previous observations where synNotch with a relatively small ECD 308 exhibited significant ligand-independent activation (10-50% of ligand-induced activation)64. 309

We also show that the size-dependent spatial segregation regulates APP cleavage and Aβ 310 production. It has been previously shown that g-secretase presenting in different subcellular 311 compartments cleaves APP into diverse Aβ isoforms70,73,76-78. Our study shows that, after the ECD 312 cleavage, cadAJ potentiates cell surface processing of APPs within the junction, yielding Aβ40 313 predominantly, while removal of cadAJ produces more Aβ42. To establish the relevance of this 314 observation to APP processing will require further investigation in a neuronal system, but our results in 315 model cell lines are consistent with the predominant secretion of Aβ from synapse, where N-cadherin 316 junctions localize79,80. 317

The spatial switch is analogous to the kinetic segregation model of T cell activation, where large 318 CD45 phosphatase is excluded from T cell receptor (TCR) immunological synaptic clefts28,32-42. However, 319 there are several distinct features of the Notch spatial switch compared to the kinetic segregation model. 320 First, unlike the immunological synapse where TCR and CD45 remain constant in size throughout 321 activation, Notch undergoes a dramatic size change during the course of cell surface activation, enabling 322 its dynamic spatial redistribution and sequential proteolysis. Second, the role of cadAJs in Notch signaling 323 is not limited to creating a physical barrier, but also plays the critical role of recruiting g-secretase to 324 facilitate processing of S2-cleaved Notch at the cell surface. Third and finally, the consequences of size-325 dependent segregation on signaling are reversed compared to the immunological synapse. While spatial 326 segregation of CD45 enables sustained TCR phosphorylation and downstream signaling, Notch 327 segregation from cadAJs inhibits signal activation. Our result extends the relevance of size-dependent 328 spatial segregation models beyond immune cells, 28,32-43, supporting the notion that the size-dependent 329 protein segregation can serve as a general mechanism for regulating a broad range of receptor signaling 330 at the cell-cell interface, including Notch and APPs. 331 Overall, a spatial switching mechanism based on size-dependent protein segregation not only 332 sheds light on the mechanism underlying the sequential proteolysis of Notch and APPs, but also may 333 extend to other receptors processed by γ-secretase. Finally, we anticipate further implications of our work 334 in other areas of research such as providing new design principles for synthetic receptors like synNotch, 335 as well as new therapeutic approaches that target Notch and APP signaling by spatial mutation in cancer 336 and neurodegenerative diseases. 337


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Acknowledgement 506 The authors thank Drs. Stephen Blacklow (Harvard U.) and C. Miller (King’s College London) for the kind 507 gifts of Notch and APP plasmids, respectively. We also thank Dr. Daniel Fletcher (UC Berkeley), Mr. Ari 508 Joffe (UC Berkeley), and Dr. Duaa Al-Rawi (Stanford U.) for helpful discussion. For reagents, technical 509 supports, and discussions we thank the Kim, Cheon, Gartner, and Jun laboratories, as well as the Nikon 510 Imaging Center and Wynton at UCSF. M.K. was supported by a Life Science Research Foundation 511 fellowship as the Shurl and Kay Curci Foundation fellow, and by Burroughs Wellcome Travel Fund. This 512 work was supported by 2020R1A2C4002533 (D.S.) and NRF-2017R1A2B3004198 (H.K.), HI17C0676 513 from Korean Ministry of Health and Welfare (H.K.), 5R01AG008200 from National Institute on Aging (NIA) 514 and the National Institute of Health (NIH) (N.K.R.), IBS-R026-D1 from IBS (H.K. and J.C.), NRF-515 2019R1A2C1085712 (Y.H.K.), the UCSF Center for Cellular Construction (an NSF Science and 516 Technology Center, no. DBI-1548297) (Z.J.G.), U01CA244109 from the National Cancer Institute (Z.J.G), 517 1R01GM112081, 1R01GM126542-01, and R35GM134948 from the National Institute of General Medical 518 Science (NIGMS) and the NIH (Y.J.), and the UCSF Program for Breakthrough Biomedical Research 519 (PBBR) funded in part by the Sandler Foundation (Y.J.). Z.J.G. is a Chan Zuckerberg BioHub Investigator. 520

Author contributions 521 M.K., K.M.S., Z.J.G., and Y.J. conceived the ideas and designed research; M.K. and K.M.S. constructed 522 plasmids, generated cell lines, and performed confocal microscopy. M.K. performed mechanogenetics, 523 truncation study, spatial mutation, immunoblot analysis, reporter cell assay, and APP experiment. K.M.S. 524 performed Notch exclusion and activation experiments, designed truncation study, wrote custom python 525 image analysis scripts. N.K. performed coarse-grained MD simulation. R.G. generated cadherin-KO cells. 526 W.R.K. performed flotillin staining and mechanogenetic experiment. M.A. and H.L. synthesized magnetic 527 nanoparticles and helped confocal imaging. J.F. and D.S. performed initial proof-of-concept experiment. 528 A.G. and N.K.R. provided anti-PS1 antibodies. H.K., Y.H.K., and J.C. oversaw and supervised CRISPR-529 Cas9 KO experiment, MD simulation, and magnetic nanoparticle synthesis, respectively. Z.J.G. oversaw 530 and supervised all spatial mapping and mutation experiments. Y.J. oversaw and supervised all aspects 531 of the experiment. M.K. and K.M.S. analyzed data. M.K., K.M.S., Z.J.G., and Y.J. wrote the manuscript. 532

Competing interests 533 The authors declare no competing interest. 534

Additional information 535

Supplementary Information is available for this paper. 536 537


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538

Figure 1. Spatial segregation of Notch receptors and ligands from cadAJs prevents their 539 interactions with γ-secretase. a, A schematic showing spatial distribution of Notch signaling 540 components at the juxtaposed cell-cell interface. b, Representative confocal fluorescence images 541 showing presenilin-1 (PS1), Notch1, and Dll1 distributions relative to E-cadherin-based cadherin junction 542 (cadAJ). (top) A maximum projection image. Scale bar, 5 µm. (bottom) z-resliced images. Scale bar, 3 543 µm. PS1 was strongly enriched at the cadAJ, whereas Notch1 and Dll1 were segregated from the cadAJ. 544 c, Line profiles quantifying fluorescence from E-cadherin (green), PS1 (cyan), Notch (red), and Dll1 545 (magenta) along a representative section of the cadAJ (a white dashed line in panel b, z-resliced images). 546 d, A schematic showing mechanogenetic interrogation of γ-secretase and Notch distribution relative to 547 the artificial cadAJs. Artificial cadAJs were formed by clustering Ecad-GFP labeled with 548 magnetofluorescent nanoparticles (MFNs) by application of an external micromagnetic tweezer (µMT). 549 DAPT was used to inhibit γ-secretase activity during mechanogenetic interrogation of Notch/γ-secretase 550 interaction in absence of the membrane juxtaposition. e, Epifluorescence images showing the formation 551 of an artificial cadAJ by mechanogenetics. After stimulation by µMT, vivid MFN and E-cadherin signals 552 at the magnetic focus were seen, indicating formation of cadAJs. Scale bar, 5 µm. f, Confocal 553 fluorescence images of E-cadherin, PS1, and Flotillin-1 (Flot1) at the artificial cadAJ. Scale bar, 2 µm. 554 Line profiles of E-cadherin, PS1 and Flot1 signals along a white dashed line. ΔI/I0 represents a fold 555 change relative to nonjunctional membrane signals. g, Confocal fluorescence images showing E-556 cadherin and Notch distributions at the artificial cadAJs after µMT application. Strong accumulation of 557 Notch signals at the artificial cadAJ was clearly seen. Line profiles of MFN, E-cadherin, and Notch signals 558 along a white dashed line. ΔI/I0 represents a fold change relative to fluorescence intensity before 559 stimulation. Scale bar, 2 µm. 560 561

562


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563

Figure 2. Size-dependent segregation of Notch at the cadAJs controls the proteolytic sequence. 564 a, A schematic to capture the spatial distribution of Notch intermediates during the cell-surface activation 565 pathway. b, Confocal z-resliced images showing Notch distribution (red) relative to cadAJ (green) from 566 the cells without Dll4 activation (i), treated with Dll4 and TAPI2 (ii), treated with Dll4 and DAPT (iii), and 567 washed out to remove DAPT inhibition (iv). Scale bar, 3 µm. c, Quantification of Notch signal enrichment 568 at the cadAJs during the activation. Notch enrichment (IIN/IOUT) is calculated as the ratio of average Notch 569 fluorescence intensity within cadAJs (IIN) and outside cadAJ (IOUT). The enrichment factor of Dil is present 570 as a control showing cadAJ-independent distribution. ****P < 0.0001, ns: non-significant, one-way 571 ordinary ANOVA followed by Tukey’s multiple comparison testing. d, Schematics of Notch variants with 572 different truncation lengths, in comparison with the cadAJ intermembrane cleft. e, Confocal fluorescence 573 images showing spatial distribution of the Notch variants (red) relative to the cadAJs (green). To prevent 574 any ligand-independent activation, cells were incubated with TAPI-2 and DAPT. (left) Maximum 575


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projections of confocal z-stacks are shown. Scale bar, 5 µm. (right) Confocal z resliced images along the 576 white dashed lines in the maximum projection images. Scale bar, 2 µm. f, Quantification of the enrichment 577 factor (IIN/IOUT) of Notch variants relative to the cadAJs. A box-plot showing binary localization of NΔEGF 578 which are defined as either excluded (yellow) or enriched (orange) is shown on the right. **P < 0.01, ***P 579 < 0.001, ns: non-significant, one-way ordinary ANOVA followed by Tukey’s multiple comparison testing. 580 g, Confocal z-resliced images showing the distribution of extracellular SNAP (purple) and intracellular 581 mCherry (red) tags of NΔEGF relative to Ecad-AJs (green) after DAPT removal. Scale bar, 3 µm. h, A 582 box-whisker plot showing Pearson correlation coefficients of extracellular-SNAP (purple) and 583 intracellular-mCherry (red) domains relative to the cadAJs before and after DAPT washout (***P < 0.001; 584 ns, not significant; n = 9 biological replicates; two-tailed unpaired Student’s t-test). i, Time series of 585 confocal z-resliced images showing the enrichment of NΔEGF (red) at the cadAJ (green) under DAPT 586 treatment (t = 0), and the dissipation during DAPT washout (t ≥ 2). Scale bar, 3 µm. j, Single-cell traces 587 showing the time-course of the decline of NΔEGF enrichment factor at the cadAJs during DAPT washout 588 (mean ± s.e.m.; n = 4 independent single-cell experiments; see Extended Data Figure 6, d-f). k, l, Western 589 blot analyses of cleaved NICD levels in the cells stably expressing NFL, NΔEGF1-25, NΔEGF, and NEXT. 590 All cells were transfected with Ecad-GFP and incubated with TAPI2 for 24 hr. β-actin levels represent the 591 loading control. A representative image of immunoblotting (k), and quantification (l) of cleaved NICD 592 levels. The average intensity of each NICD band relative to respective β-actin band was quantified and 593 then normalized to that of NEXT (mean ± s.d.; n = 4 biological replicates; *P < 0.05, ****P < 0.0001, one-594 way ANOVA followed by Tukey’s multiple comparison test). (c, f, h) Boxes and whiskers indicate the 595 interquartile and the full ranges, respectively. Colored lines and (+) marks indicate median and mean, 596 respectively. 597

598


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599

Figure 3. Spatial mutation directs Notch activation, regardless of ligand presentation or S2 600 cleavage. a, A schematic describing DNA-mediated crosslinking strategy to enhance NΔEGF localization 601 at the cadAJ. b, Confocal z-resliced images showing intense NΔEGF fluorescence (red) enriched at the 602 cadAJ (green) after the DNA crosslinking. Scale bar, 3 µm. Quantification of enrichment (IIN/IOUT) without 603 (n = 33) with DNA crosslinker treatment (n = 29), indicating an increase in enrichment after the 604 crosslinking (*P < 0.05; two-tailed Mann-Whitney-Wilcoxon test). c, Western blot analyses showing 605 increased S3-cleaved NICD levels in the NΔEGF cells treated with the DNA crosslinker. (top) A 606 representative gel image showing immunoblotting for NICD and β-actin. (bottom) Quantification of 607 cleaved NICD levels. The average intensity of NICD band was normalized to that of β-actin band in each 608 sample. (mean ± s.d.; ** P < 0.01; n = 5 biological replicates; ordinary one-way ANOVA). d, Spatial 609 mutation of NEXT via chemical ligation of macromolecular pendants (denoted as P). 10 µM of BG-610 modified polymers or proteins were conjugated to the extracellular SNAP tag of NEXT. Cartoons 611 depicting shape and hydrodynamic size of different pendants are shown. Confocal images showing size-612 dependent spatial mutation of NEXT (red) at the cadAJs (green). The top row shows maximum projection 613 images of the cells treated with the indicated pendants. Scale bar, 5 µm. The middle row shows confocal 614 z resliced images along the white dashed lines in the maximum projection images. Yellow and green 615 arrowheads indicate the cadAJs enriches with and those that excludes Notch, respectively. Scale bar, 3 616


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µm. The bottom row shows line profiles quantifying fluorescence signals from NEXT (red) and E-cadherin 617 (green) along the white lines in the z-resliced images. Images and line profiles are representative of n ≥ 618 15 biological replicates. e, Quantification in IIN/IOUT of NEXT with macromolecular pendants (n ≥ 15). f, 619 Confocal fluorescence images of nuclear mCherry signals resulting from nuclear translocation of cleaved 620 NICD (red) in NEXT cells treated with macromolecular pendants. DAPI signals (blue) indicate cell nucleus. 621 Scale bar, 5 µm. g, Quantification of nuclear mCherry fluorescence for NEXT cells treated with the 622 pendants. Cells expressing NFL were used as a negative control. Data shown are from single cells (n ≥ 623 180) identified in large-area confocal images. h, A plot representing the NICD level of various Notch 624 variants as a function of the enrichment factor (IIN/IOUT, mean ± s.e.m.; n ≥ 15 biological replicates). All 625 Notch variants with different truncation length, DNA crosslinking, or pendant addition used in Figures 2 626 and 3 were included. The Notch variants showing exclusion, mixed distribution, and enrichment were 627 indicated with red, yellow, and green background colors, respectively (median ± s.e.m.; n ≥ 4 for western 628 blot for NICD levels; n ≥ 180 for nuclear mCherry fluorescence). (e, g) Boxes and whiskers indicate the 629 interquartile and the full ranges, respectively. Black lines and (+) marks indicate median and mean, 630 respectively. **P < 0.01; ****P < 0.001; ns, not significant; ordinary one-way ANOVA followed by Tukey’s 631 multiple comparison. 632

633


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634

Figure 4. The cadAJ-mediated spatial switch regulates Notch and APP signaling. a, Representative 635 fluorescence images showing Notch activation in U2OS SNAP-NFL-Gal4 reporter cell lines in different 636 cellular environments: Group of cells (left), solitary cells with no prior contact (middle), and solitary cells 637 plated on an Ecad-Fc coated substrate (right). All cells were plated on Dll4-Fc coated substrates. Scale 638 bars, 20 µm (see supplementary video 1, 2). b, Quantification of Notch activation by measuring H2B-639 mCherry fluorescence changes (intensity at t = 16 hr minus intensity at t = 0 hr) in cells within a group (n 640 = 152), solitary cells (n = 50). c, Quantification of Notch activation in solitary cells cultured on an Ecad-641 Fc-coated substrate (n = 27) and those cultured without ligands (n = 27). d, Representative confocal 642 images of H2B-mCherry fluorescence in U2OS SNAP-NFL-Gal4 reporter cells (wt), E-cadherin knockout 643 cells (Ecad-KO), Ecad-KO cells with recombinant E-cadherin transfection (Ecad-KO + Ecad), and Ecad-644 KO cells with N-cadherin transfection (Ecad-KO + Ncad). Cytosol labeled with CMFDA dye was shown 645 for wt and Ecad-KO cells. E-cadherin and N-cadherin were shown for Ecad-KO + Ecad and Ecad-KO + 646 Ncad cells. Scale bar, 100 µm. e, Quantification of Notch activation in the wt, Ecad-KO, Ecad-KO + Ecad, 647 and Ecad-KO + Ncad cells. Data shown are from n ≥ 50 single cells analyzed from two independent 648 experiments. f, (left) Confocal fluorescence maximum projection (left) and z-resliced images of U2OS 649 cells co-expressing Ncad-mCherry (green) and APP-EGFP (red). To capture the APP intermediates, cells 650 were cultured with a combination of α-, β-, and g-secretase inhibitors. Scale bars, 10 µm (maximum 651 projection) & 3 µm (inset). g, Aβ42/Aβ40 ratio measured by ELISA in wild-type cells or CDH1/2 KO cells 652 (mean ± SEM, n = 3, *P< 0.05, two-tailed paired Student’s t test). (b, c, and e) Boxes and whiskers 653 indicate the interquartile and full ranges, respectively. Black lines and (+) marks indicate median and 654 mean, respectively. **P < 0.01; ****P < 0.001; ns, not significant; unpaired two-tailed t test in (b) and (c); 655 ordinary one-way ANOVA followed by Tukey’s multiple comparison in (e). 656

657


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658 659 Extended Data Figure 1. Distribution of ADAM10 and γ-secretase at the cell-cell interface. a, 660 Representative confocal fluorescence images showing ADAM10 distribution relative to E-cadherin-based 661 adherens junction (cadAJ). U2OS cells were transfected with Ecad-GFP (green), and stained with 662 ADAM10 antibody (red). Z-resliced images were reconstructed along the white dashed lines. Scale bars, 663 5 µm and 3 µm for maximum intensity projection and z-resliced images, respectively. b, Additional 664 immunofluorescence confocal images showing endogenous presenilin-1 (PS1, magenta) distribution 665 relative to cadAJs (green). Paxillin was also imaged as a negative control (blue) Scale bars, 10 µm, 3 666 µm, and 3 µm for maximum intensity projection, zoomed-in, z-resliced images, respectively. c, Cross-667 correlation analysis of PS1, ADAM10, and Notch distributions over E-cadherin. PS1 fluorescence 668 intensities exhibited strong positive correlation with cadAJs, while Notch showed nearly zero correlation. 669 ADAM10 showed a weakly positive correlation over cadAJs. The cadAJs were set as x = 0 in the analyses. 670 The shading along the curves represent error bars (s.e.m.; n ≥ 9). 671


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672 673

Extended Data Figure 2. Notch exclusion from cadAJs is consistent for several cadherin family 674 proteins, host cell types, and cell polarization states. A series of cell lines including U2OS, MDCK, 675 and HUVEC were imaged. a, b, U2OS (a) and MDCK (b) cells were co-transfected with plasmids 676 encoding Ecad-GFP (green) as well as SNAP-NFL-mCherry. Notch receptors were labeled with BG-677 Alexafluor647 (magenta). Line profiles show fluorescence signal intensities along the white lines. Scale 678 bars 10 µm, 5 µm, and 2 µm for maximum intensity projection, zoomed-in, and z-resliced images, 679 respectively. c, HUVECs were transfected with SNAP-NF-mCherry and labeled with SNAP surface 647. 680 VE-cadherin was immunostained with an anti-vascular endothelial cadherin (VE-cad) antibody. Line 681 profiles show fluorescence signal intensities along the white lines. Scale bars 5 µm and 2 µm for 682 maximum intensity projection and z-resliced images, respectively. d, Polarized MDCK cells grown on a 683 transwell filter. Scale marked every 5 µm for 3D construction and Scale bar is 2 µm for z-resliced images. 684 e, A schematic representation of Notch localization in a polarized epithelial cell. 685


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686 687 Extended Data Figure 3. Quantitative analysis of PS1, Notch, Dll distribution relative to cadAJs 688 and additional confocal images representing their spatial distribution. a, Confocal images of U2OS 689 cells co-expressing Ecad-GFP (green) and SNAP-NFL-mCherry (red). Cells were also immunostained 690 with anti-PS1 antibodies (cyan). Line profiles show fluorescence signal intensities along the white lines. 691 Scale bars, 10 µm, 2 µm, and 2 µm for maximum intensity projection, zoomed-in, and z-resliced images, 692 respectively. b, Paired analysis of Manders’ overlap coefficients of E-cadherin and Notch signals over 693 PS1 in multiple cells (n = 9). ***P < 0.001. c, Box-whisker plots showing Manders’ overlap coefficients 694 (MOCs) of PS1 (cyan), Notch (red), and Dll1 (purple) relative to cadAJs. Each dot represents the MOC 695 of a selected cadAJ. Boxes and whiskers denote the inner-quartile and full ranges. Colored lines and (+) 696 marks indicate median and mean, respectively (****P < 0.0001; ns, not significant; n ≥ 11; ordinary one-697 way ANOVA followed by Tukey’s multiple comparison test). d, Confocal fluorescence images of U2OS 698 cells co-expressing Ecad-GFP (green) and Halo-Dll1. Halo-Dll1 was labeled with Halo-660 dyes 699 (magenta). Scale bars, 5 µm, 1 µm, and 1 µm for maximum intensity projection, zoomed-in, and z-resliced 700 images, respectively. 701


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702

Extended Data Figure 4. Interrogation of the mechanism underlying gg-secretase recruitment into 703 cadAJs through mechanogenetics, cell-cell cadAJ spatial mapping, molecular dynamics 704 simulation, and cholesterol depletion experiments. a, b, Additional mechanogenetic experiments 705 showing PS1 and Flot1 localizations at cadAJs. E-cadherin and Flot1 were labeled with fluorescent tags. 706 Endogenous PS1 was immunostained after fixation. Scale bar, 20 µm. c, d, Representative confocal 707 fluorescence images showing the PS1 and Flot1 distribution relative to native cell-cell cadAJs. (left) A 708


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maximum projection image of merged channels. Scale bar, 10 µm. (center) Magnified images showing 709 greater details of the boxed region. Scale bar, 2 µm. (right) Z-resliced images showing the sections of 710 the cadAJs. Scale bar, 2 µm. Line profiles of fluorescence signals from E-cadherin, PS1, and Flot1 along 711 the white dashed lines in the z-resliced images. e, Cross-correlation analysis of E-cadherin and PS1 over 712 Flot1. Both Flot1 and PS1 fluorescence intensities exhibited strong positive correlation with the cadAJ. 713 The shades along the curves represent error bars (s.e.m.; n = 7). f, Manders’ overlap coefficients of E-714 cadherin and Flot-1 signals over PS1. Two-tailed paired Student’s t test, ns: not significant. g, Quantitative 715 assessment of Flot1 and Notch colocalization with E-cadherin. Pearson correlation coefficient is shown. 716 Each circle is the score of a single cadAJ. Overlaid are box and whisker plots. **** P

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731

732 733 Extended Data Figure 5. Additional confocal images representing recruitment of Notch receptors 734 to the artificial cadAJ. a, Pre-stimulation images of a subcellular locus of the cell shown in Fig. 1g. Initial 735 dim MFN (magenta), E-cadherin (green), and Notch (red) signals were evenly distributed. Scale bar, 2 736 µm. b, c, Additional artificial cadAJs showing Notch recruitment. Time-lapse images (c) were acquired 737 before micromagnetic tweezer (µMT) stimulation and then at 10, 20, 30 minutes of the µMT application. 738 Gradual MFN and E-cadherin clustering were clearly seen, followed by Notch accumulation to the cadAJ. 739 Scale bar, 2 µm. d. Kinetics of signal enrichments at the artificial cadAJ shown in the panel c. 740

741


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742

743 Extended Data Figure 6. Confocal images showing the gradual release of NICD from S2-cleaved 744 Notch concentrated within cadAJs. a, Representative confocal images and enrichment factors (IIN/IOUT) 745 of Dil membrane staining dye distribution relative to cadAJs. b, Representative time-course z-resliced 746 images showing S2-cleaved Notch at cadAJs as a function of time after DAPT removal. The NICD signal 747 (red) at the cadAJ gradually decreases, indicating NICD release. Images shown here are not from 748 identical cells, but represent a general trend of NICD signal at cadAJs for each time point. c, 749 Quantification IIN/IOUT ratio as a function of time after DAPT washout. d-f, Time traces of NICD release 750 from U2OS cells expressing NΔEGF (red) and Ecad-GFP (green). d, Cells showing strong NΔEGF 751 enrichment at cadAJs under DAPT treatment were chosen. DAPT was removed while keeping TAPI2 752 concentration constant and time series confocal images were acquired at 0, 2, 4, and 6 hrs from DAPT 753 removal. The white arrows indicate the locations of two representative cadAJs shown and analyzed in 754 Fig. 2i, j. e, Single-cell traces (n = 4) of enrichment factor. f, Quantification of changes in NICD signal 755 from these four cells at the cadAJs and non-cadAJ membrane, at t = 0 (green, before washout) and t = 756 6 hr (red, after DAPT wash-out). CadAJs and non-cadAJ membrane were detected based on thresholding 757 and automatic segmentation using the custom-built script. Average mCherry fluorescence signals from 758 cadAJs and non-cadAJ membranes were also measured using the same script. Intracellular-mCherry 759 signals significantly decreased at the cadAJs, but not at non-cadAJ membranes (* P < 0.05, ns: non-760 significant, paired two-tailed Student’s t-test, n = 4). Scale bars, 5 µm. 761


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762 763

Extended Data Figure 7. Spatial mutation of NΔEGF using DNA-mediated crosslinking. a. A 764 representative western blot of lysate from cells after 2 hr incubation with or without DNA crosslinkers. 765 The blot was labelled with anti-SNAP and anti-β-actin antibodies. The expected mass of NΔEGF and its 766 complex with the Notch construct and E-cadherin forming a heterodimer is 90 kD and 230 kD, respectively. 767 β-actin detection was used to assess protein loading. Predicted bands representing Notch-E-cadherin 768 heterodimers (heterodimer) and SNAP-NΔEGF-mCherry monomers are indicated with solid lines. 769 Molecular weights are indicated on the left. b. Representative maximum intensity projection confocal 770 images showing the distribution of NΔEGF relative to the cadAJs after crosslinking. Cells were treated 771 with or without DAPT. Scale bar, 10 µm. c, d, Removal of DAPT induced S3 cleavage of NΔEGF at the 772 cadAJs. c, Single-cell confocal images showing intracellular-mCherry signals at the cadAJ under DNA 773 and DAPT treatment (left) and after washing out DAPT (right). Removing DAPT elicited a significant 774 reduction in mCherry signal intensity from the cadAJ. Scale bar, 5 µm. d. Paired analysis of multiple cells 775 expressing NΔEGF (n = 6) in enrichment factor (IIN/IOUT) after DAPT washout. Each dot represents IIN/IOUT 776 value before and after DAPT washout from a single cell. Each line corresponds to the IIN/IOUT changes 777 before and after DAPT washout in a same single cell (**P

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780 781 Extended Data Figure 8. Spatial mutation of NEXT using chemical ligation of macromolecular 782 pendants. a, Dynamic light scattering spectra of BG-modified macromolecules used in the experiment 783 to induce spatial mutation of NEXT in Fig. 3d-g. b, Larger area (1 x 1 mm) confocal fluorescence images 784 shown in Fig. 3f. Scale bar, 200 µm. 785


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786 Extended Data Figure 9. Generation of U2OS SNAP-NFL-Gal4 fluorescence reporter cell lines 787 lacking E- and N-cadherins via CRISPR/Cas9. a. Schematic representation of human CDH1 gene 788 structure and targeted segmental deletion sites. The sixteen exons are shown in orange boxes (E1-E16). 789 Red arrowheads indicate the sgRNA-binding sites (E1LT1, E2RT1, E13LT1, E13LT2, E14RT1 and 790 E14RT2). The targeted segmental deletion of 940bp and 4.6kb is shown with a black line with red arrow 791 tips, respectively. Purple arrows represent PCR primers used for the T7E1 assay and detection of alleles 792 with targeted deletions, respectively. b, c, T7E1 assays to evaluate the efficiency of each sgRNAs. U2OS 793 SNAP-NFL-Gal4 reporter cells were analyzed after transfection with plasmids encoding Cas9 and each 794 sgRNA for CDH1 (b) and CDH2 (c). Mutation frequencies (indel [%]) were calculated from the band 795 intensities. Untransfected cells were used as controls (CONT). The red arrows and asterisk indicate the 796 uncleaved and expected cleaved position of DNA bands generated by cleavage by T7E1, respectively. 797 M: Marker lane. d, Identification of clones containing the entire segmental deletion in both target alleles 798 for selected clones. The wild-type and deleted alleles were detected by PCR using different primer pairs 799 at exon12 and 13 target regions, respectively. The black arrows indicate the expected positions of the 800 amplicons with wild type allele or allele with deletions. The red arrows indicate the candidate clones 801


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containing the entire segmental deletion in both target alleles. e, Sanger sequencing of the CDH1 wild-802 type (wt) allele and alleles containing deletions of the segmental CDH1 gene. Cas9 recognition sites are 803 shown in red and the protospacer adjacent motif (PAM) sequences are shown in blue bold characters. 804 Dashes indicate deleted bases. The number of occurrences is shown in the first set of parentheses (e.g., 805 X6, X3 and X6 indicate how many times each sequence was observed). The cleavage sites are indicated 806 by red arrows. The sequence length of the consequent large deletion is shown in the second set of 807 parentheses. f, CDH1 mRNA expression levels in CDH1 KO clone #3 and #4 were determined using 808 qRT-PCR. CDH1 expression levels in the selected clones containing a segmental deletion were 809 quantified relative to CDH1 mRNA levels of the wild-type U2OS SNAP-NFL-Gal4 cells. Clone #3 was used 810 for subsequent experiments. g, Western blot analyses of cleaved NICD levels in the U2OS SNAP-Notch-811 Gal4 reporter cells (wt), Ecad-KO, and Ecad-KO cells with recombinant E-cadherin transfection (Ecad-812 KO + Ecad). (top) A representative image of immunoblotting. (bottom) Quantification of cleaved NICD 813 levels. The average intensity of NICD bands relative to β-actin bands was quantified and then normalized 814 to that of Ecad-KO cells (mean ± s.d.; ***P < 0.001; n = 5 biological replicates; one-way ANOVA followed 815 by Tukey’s multiple comparison test). h, PCR-based evaluation of targeted segmental deletions in double 816 KO (CDH1 and CDH2) single-cell derived clones. U2OS SNAP-NFL-Gal4 cells were transfected with 817 plasmids encoding Cas9 and two sets of sgRNA pairs, each targeting CDH1 and CDH2, respectively. 818 The red arrow indicates the expected position of the amplicon from the region containing the segmental 819 targeted deletion M: Marker lane. i, To obtain the bi-allelic KO clones, we validated bi-allelic deletion in 820 both CDH1 and CDH2 genes from the indicated clones. The red arrows indicate the expected positions 821 of the amplicons with wild type allele or allele with deletions. Clone #11 (red color), showing bi-allelic 822 deletion of both CDH1 and CHD2, was used for further subsequent experiments. M: Marker lane. 823

824


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Extended Data Table 1. Cell lines used in this study. 825 Cell line Stable

expression*,** CRISPR-KO

Transient expression 1*,**

Transient expression 2*,**

Figure

Plain U2OS

Halo-Dll1 Ecad-GFP 1(b-d) EDF# 3a

Ecad-GFP Flot1-Halo 1(e-g) EDF 3(d-f)

SNAP-Ecad-GFP Flot1-Halo 1j EDF 5(a-d)

Ecad-GFP EDF 1(a-c) APP-GFP SNAP-Ncad 4f

T-rex U2OS (doxycycline inducible)

SNAP-NFL-mCherry (NFL)

Ecad-GFP 1(b-d) 2(b, c) 2(k, l) 3h EDF 2a EDF 3(b,c) EDF 7(a,b)

Halo-Ecad-GFP 1(k, l) EDF 6(a,b) EDF 8(a-d)

SNAP-NΔEGF1-25-mCherry (NΔEGF1-25)

Ecad-GFP 2(e, f) 2(k, l) 3h

SNAP-NΔEGF-mCherry (NΔEGF)

Ecad-GFP 2(e-l) 3h EDF 7(c-e)

Halo-Ecad-GFP 3(a-c) 3h

SNAP-NEXT-mCherry (NEXT)

Ecad-GFP 2(e, f) 2(k, l) 3(d-h) EDF 9b

SNAP-NFL-Gal4 & UAS-H2B-mCherry

4(a-e) Ecad Ecad-GFP 4(d, e)

Ncad-GFP 4(d, e) SNAP-NFL-Gal4 & UAS-H2B-mCherry (uninduced)

APP-GFP SNAP-Ncad 4g Ecad & Ncad

APP-GFP 4g

MDCK SNAP-NFL-mC Ecad-GFP EDF 2(b,d) HUVEC SNAP-NFL-mC EDF 2c

826 *X-Y represents a fusion protein of X with Y. 827 **X-Y-Z represents a Y protein fused with X- and Z- tag proteins at its N- and C-termini, respectively. 828 #Extended Data Figure 829 830 Abbreviations: 831 Notch receptors and ligands 832 NFL: full length Notch 833 NΔEGF1-25: Notch with EGF domain truncation from 1-25. 834 NΔEGF: Notch with complete EGF repeat domain truncation. 835 NEXT: Notch with extracellular domain truncation 836 Dll1: Delta like ligand 1 837


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838 Cadherins 839 Ecad: epithelial (E) cadherin 840 Ncad: neural (N) cadhrin 841 VEcad: vascular endothelial cadherin 842 843 Amyloid precursor proteins 844 APP: amyloid precursor proteins with deletion of YENPTY motifs 845 846 Self-labeling and fluorescent protein tags 847 SNAP: SNAP tag 848 Halo: Halo tag 849 GFP: green fluorescent protein 850 851 Others 852 Flot1: flotillin 1 853 H2B: histone 2B 854 UAS: upstream activation sequence 855 856 857 858 859 860 861

862


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METHODS 863 Cloning, cell culture, transfection, and cell line generation 864 Cloning 865 Plasmid constructs used in this study are listed in Extended Data Table 1. All constructs used in this 866 paper were assembled using standard restriction enzyme-based cloning, in fusion cloning, and/or Gibson 867 isothermal assembly. The maps, sequences, and construction details of all plasmids are available upon 868 request. All constructs were sequenced to confirm mutation. Complete details of all cloning procedures 869 are available upon request. 870 Flag-human Notch1 (NFL)-Gal4 and pGF1-UAS-H2B-mCherry were gifts from S. Blacklow (Harvard 871 University). Flag-human NFL-Gal4 was provided in a Tet-ON Flp-IN vector (pcDNA5). SNAP-NFL-mCherry 872 and SNAP-NFL-Gal4 were constructed as previously reported1. All Notch1 variants with partial or full 873 extracellular domain truncation were constructed by linearizing and amplifying SNAP-hN1-mCherry 874 vector via inverse PCR while omitting the sequence corresponding the ECD truncation. Notch 875 ectodomain sequences of amino acid 23-981, 23-1426, 23-1709 were deleted for SNAP-ΔEGF1-25-876 mCherry. SNAP-ΔEGF-mCherry. and SNAP-NEXT-mCherry. respectively. 877 Ecad-GFP was purchased from Addgene (Addgene plasmid # 28009; 878 http://n2t.net/addgene:28009). SNAP-Ecad-GFP and Halo-Ecad-GFP were constructed first by 879 linearizing and amplifying Ecad-EGFP vector via inverse PCR. SNAP- and Halo-tags were then inserted 880 in

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10 11 12 13 14 15 16 17 18 · 2020. 6. 28. · 9 1Department of Otolaryngology, University of California, San Fr ancisco, CA, USA 10 2Department of Pharmaceutical Chemistry, University